Ucp4

ABSTRACT

The present invention is directed to a novel polypeptide, designated in the present application as “UCP4” (SEQ ID NO: 1), having homology to certain human uncoupling proteins (“UCPs”) and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention, and methods for producing the polypeptides of the present invention.

RELATED APPLICATIONS

This is a non-provisional application filed under 37 CFR 1.53(b)claiming priority to and is a continuation of application Ser. No.11/265,966, filed Nov. 3, 2005, now U.S. Pat. No. 8,067,229 issued Nov.29, 2011, which is a continuation of application Ser. No. 09/397,342,filed Sep. 15, 1999, now abandoned, which claims the benefit of U.S.Provisional Application Nos. 60/129,674, filed Apr. 16, 1999,60/114,223, filed Dec. 30, 1998, and 60/101,279, filed Sep. 22, 1998,the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA having homology to certain human uncouplingproteins, and to the recombinant production of novel polypeptides,designated herein as “uncoupling protein 4” or “UCP4.”

BACKGROUND OF THE INVENTION

Uncoupling proteins or “UCPs”, believed to play a role in the metabolicprocess, have been reported in the literature. UCPs were first found anddescribed in the brown fat cells of hibernating animals, such as bears.UCPs were believed to help such hibernators and other cold-weatheradapted animals maintain core body temperatures in cold weather byraising their body's resting metabolic rate. Because humans possessrelatively small quantities of brown adipose tissue, UCPs wereoriginally thought to play a minor role in human metabolism.

Several different human uncoupling proteins have now been described.[See, generally, Gura, Science, 280:1369-1370 (1998)]. The humanuncoupling protein referred to as UCP1 was identified by Nicholls et al.Nicholls et al. showed that the inner membrane of brown fat cellmitochondria was very permeable to proteins, and the investigatorstraced the observed permeability to a protein, called UCP1, in themitochondrial membrane. Nicholls et al. reported that the UCP1, bycreating such permeability, reduced the number of ATPs that can be madefrom a food source, thus raising body metabolic rate and generatingheat. [Nicholls et al., Physiol. Rev., 64, 1-64 (1984)].

It was later found that UCP1 is indeed expressed only in brown adiposetissue [Bouillaud et al., Proc. Natl. Acad. Sci., 82:445-448 (1985);Jacobsson et al., J. Biol. Chem., 260:16250-16254 (1985)]. Geneticmapping studies have shown that the human UCP1 gene is located onchromosome 4. [Cassard et al., J. Cell. Biochem., 43:255-264 (1990)].

Another human UCP, referred to as UCPH or UCP2, has also been described.[Gimeno et al., Diabetes, 46:900-906 (1997); Fleury et al., Nat. Genet.,15:269-272 (1997); Boss et al., FEBS Letters, 408:39-42 (1997); seealso, Wolf, Nutr. Rev., 55:178-179 (1997)]. Fleury et al. teach that theUCP2 protein has 59% amino acid identity to UCP1, and that UCP2 maps toregions of human chromosome 11 which have been linked tohyperinsulinaemia and obesity. [Fleury et al., supra]. It has also beenreported that UCP2 is expressed in a variety of adult tissues, such asbrain and muscle and fat cells. [Gimeno et al., supra, and Fleury etal., supra].

A third human UCP, UCP3, was recently described in Boss et al., supra;Vidal-Puig et al., Biochem. Biophys. Res. Comm., 235:79-82 (1997);Solanes et al., J. Biol. Chem., 272:25433-25436 (1997); and Gong et al.,J. Biol. Chem., 272:24129-24132 (1997). [See also Great Britain PatentNo. 9716886]. Solanes et al. report that unlike UCP1 and UCP2, UCP3 isexpressed preferentially in human skeletal muscle, and that the UCP3gene maps to human chromosome 11, adjacent to the UCP2 gene. [Solanes etal., supra]. Gong et al. describe that the UCP3 expression can beregulated by known thermogenic stimuli, such as thyroid hormone,beta3-andrenergic agonists and leptin. [Gong et al., supra].

SUMMARY OF THE INVENTION

A cDNA clone (DNA 77568-1626) has been identified, having certainhomologies to some known human uncoupling proteins, that encodes a novelpolypeptide, designated in the present application as “UCP4.”

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a UCP4 polypeptide.

In one aspect, the isolated nucleic acid comprises DNA having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to (a) a DNA moleculeencoding a UCP4 polypeptide comprising the sequence of amino acidresidues from about 1 to about 323, inclusive of FIG. 1 (SEQ ID NO:1),or (b) the complement of the DNA molecule of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a UCP4 polypeptide comprising DNA hybridizing to thecomplement of the nucleic acid between about nucleotides 40 and about1011 inclusive, of FIG. 2 (SEQ ID NO: 2). Preferably, hybridizationoccurs under stringent hybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA having at least about 80% sequence identity,preferably at least about 85% sequence identity, more preferably atleast about 90% sequence identity, most preferably at least about 95%sequence identity to (a) a DNA molecule encoding the same maturepolypeptide encoded by the cDNA in ATCC Deposit No. 203134, or (b) thecomplement of the DNA molecule of (a). In a preferred embodiment, thenucleic acid comprises a DNA encoding the same mature polypeptideencoded by the cDNA in ATCC Deposit No. 203134.

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding a polypeptide having at leastabout 80% sequence identity, preferably at least about 85% sequenceidentity, more preferably at least about 90% sequence identity, mostpreferably at least about 95% sequence identity to the sequence of aminoacid residues from about 1 to about 323, inclusive of FIG. 1 (SEQ IDNO:1), or the complement of the DNA of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1to about 323, inclusive of FIG. 1 (SEQ ID NO:1), or (b) the complementof the DNA of (a).

Further embodiments of the invention are directed to fragments of theUCP4 coding sequence, which are sufficiently long to be used ashybridization probes. Preferably, such fragments contain at least about20 to about 80 consecutive bases included in the sequence of FIG. 2 (SEQID NO:2). Optionally, such fragments include the N-terminus or theC-terminus of the sequence of FIG. 2 (SEQ ID NO:2).

In another embodiment, the invention provides a vector comprising DNAencoding UCP4 or its variants. The vector may comprise any of theisolated nucleic acid molecules hereinabove defined.

A host cell comprising such a vector is also provided. By way ofexample, the host cells may be CHO cells, E. coli, or yeast. A processfor producing UCP4 polypeptides is further provided and comprisesculturing host cells under conditions suitable for expression of UCP4and recovering UCP4 from the cell culture.

In another embodiment, the invention provides isolated UCP4 polypeptideencoded by any of the isolated nucleic acid sequences hereinabovedefined.

In a specific aspect, the invention provides isolated native sequenceUCP4 polypeptide, which in one embodiment, includes an amino acidsequence comprising residues 1 to 323 of FIG. 1 (SEQ ID NO:1).

In another aspect, the invention concerns an isolated UCP4 topolypeptide, comprising an amino acid sequence having at least about 80%sequence identity, preferably at least about 85% sequence identity, morepreferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to the sequence of amino acid residues1 to about 323, inclusive of FIG. 1 (SEQ ID NO:1).

In a further aspect, the invention concerns an isolated UCP4polypeptide, comprising an amino acid sequence scoring at least about80% positives, preferably at least about 85% positives, more preferablyat least about 90% positives, most preferably at least about 95%positives when compared with the amino acid sequence of residues 1 to323 of FIG. 1 (SEQ ID NO:1).

In yet another aspect, the invention concerns an isolated UCP4polypeptide, comprising the sequence of amino acid residues 1 to about323, inclusive of FIG. 1 (SEQ ID NO:1), or a fragment thereof.sufficient to, for instance, provide a binding site for an anti-UCP4antibody. Preferably, the UCP4 fragment retains at least one biologicalactivity of a native UCP4 polypeptide.

In a still further aspect, the invention provides a polypeptide producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a UCP4 polypeptide having the sequence ofamino acid residues from about 1 to about 323, inclusive of FIG. 1 (SEQID NO: 1), or (b) the complement of the DNA molecule of (a), and if thetest DNA molecule has at least about an 80% sequence identity,preferably at least about an 85% sequence identity, more preferably atleast about a 90% sequence identity, most preferably at least about a95% sequence identity to (a) or (b), (ii) culturing a host cellcomprising the test DNA molecule under conditions suitable forexpression of the polypeptide, and (iii) recovering the polypeptide fromthe cell culture.

In another embodiment, the invention provides chimeric moleculescomprising a UCP4 polypeptide fused to a heterologous polypeptide oramino acid sequence. An example of such a chimeric molecule comprises aUCP4 polypeptide fused to an epitope tag sequence or a Fc region of animmunoglobulin.

In another embodiment, the invention provides an antibody whichspecifically binds to UCP4 polypeptide. Optionally, the antibody is amonoclonal antibody.

In yet another embodiment, the invention concerns agonists andantagonists of a native UCP4 polypeptide. In a particular embodiment,the agonist or antagonist is an anti-UCP4 antibody.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists of a native UCP4 polypeptide, comprisingcontacting the native UCP4 polypeptide with a candidate molecule andmonitoring the desired activity. The invention also provides therapeuticmethods and diagnostic methods using UCP4.

In a still further embodiment, the invention concerns a compositioncomprising a UCP4 polypeptide, or an agonist or antagonist ashereinabove defined, in combination with a carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the derived amino acid sequence of a native sequence UCP4SEQ ID NO: 1. FIG. 2 shows the nucleotide sequence of a cDNA encodingnative sequence UCP4 SEQ ID NO: 2.

FIGS. 3A-3B shows an amino acid sequence alignment of UCP4 SEQ ID NO: 1with other known uncoupling proteins, UCP1 (SEQ ID NO:16), UCP2 (SEQ IDNO:17), and UCP3 (SEQ ID NO:18). The six putative transmembrane domainsare shown and are underlined (and labeled I to VI, respectively). Theasterisks (*) shown below the protein sequence indicate three (3)putative mitochondrial carrier protein motifs. A putative nucleotidebinding domain is double underlined.

FIGS. 4A-4H show the results of Northern blot analysis. Human adulttissues and brain tissues (Clontech), in addition to peripheral bloodleukocytes (PBLs), cancer cells, and fetal tissues, were probed withUCP4 cDNA. The figures illustrate that the UCP4 transcript was detectedin human brain tissues, spinal cord, medulla, corpus callosum, andsubstantia nigra.

FIGS. 5A-5B show the results of in vitro assays conducted to determinethe effects of UCP4 expression on mitochondrial membrane potential.

FIGS. 6A-6F show the results of in vitro assays conducted to determinethe subcellular localization of UCP4.

FIG. 7 shows a “from DNA” sequence assembled from selected EST sequencesSEQ ID NO: 5.

FIGS. 8A-8C show the results of in vitro assays conducted to determinethe effect of food consumption on the expression of UCP4 mRNA.

FIGS. 9A-9D show the results of in vitro assays conducted to determinethe effect of fat consumption on the expression of UCP4 mRNA.

FIGS. 10A-10G show the results of in vitro assays conducted to determinethe effect of temperature stress on the expression of UCP4 mRNA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The terms “UCP4 polypeptide”, “UCP4 protein” and “UCP4” when used hereinencompass native sequence UCP4 and UCP4 variants (which are furtherdefined herein). The UCP4 may be isolated from a variety of sources,such as from human tissue types or from another source, or prepared byrecombinant and/or synthetic methods.

A “native sequence UCP4” comprises a polypeptide having the same aminoacid sequence as a UCP4 derived from nature. Such native sequence UCP4can be isolated from nature or can be produced by recombinant and/orsynthetic means. The term “native sequence UCP4” specificallyencompasses naturally-occurring truncated or soluble forms,naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the UCP4. In one embodimentof the invention, the native sequence UCP4 is a mature or full-lengthnative sequence UCP4 comprising amino acids 1 to 323 of FIG. 1 (SEQ IDNO:1).

“UCP4 variant” means anything other than a native sequence UCP4, andincludes UCP4 having at least about 80% amino acid sequence identitywith the amino acid sequence comprising residues 1 to 323 of the UCP4polypeptide sequence shown in FIG. 1 (SEQ ID NO:1). Such UCP4 variantsinclude, for instance, UCP4 polypeptides wherein one or more amino acidresidues are added, or deleted, at the N- or C-terminus, as well aswithin one or more internal domains, of the sequence of FIG. 1 (SEQ IDNO:1). Ordinarily, a UCP4 variant will have at least about 80% aminoacid sequence identity, more preferably at least about 85% amino acidsequence identity, even more preferably at least about 90% amino acidsequence identity, and most preferably at least about 95% sequenceidentity with the amino acid sequence comprising residues 1 to 323 ofFIG. 1 (SEQ ID NO:1).

“Percent (%) amino acid sequence identity” with respect to the UCP4sequences identified herein is defined as the percentage of amino acidresidues in a candidate sequence that are identical with the amino acidresidues in the UCP4 sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. % identity can be determined by WU-BLAST-2,obtained from [Altschul et al., Methods in Enzymology, 266: 460-480(1996)]. WU-BLAST-2 uses several search parameters, most of which areset to the default values. The adjustable parameters are set with thefollowing values: overlap span=1, overlap fraction=0.125, word threshold(T)=11. The HSP S and HSP S2 parameters are dynamic values and areestablished by the program itself depending upon the composition of theparticular sequence and composition of the particular database againstwhich the sequence of interest is being searched; however, the valuesmay be adjusted to increase sensitivity. A % amino acid sequenceidentity value is determined by the number of matching identicalresidues divided by the total number of residues of the “longer”sequence in the aligned region. The “longer” sequence is the one havingthe most actual residues in the aligned region (gaps introduced byWU-Blast-2 to maximize the alignment score are ignored).

The term “positives”, in the context of sequence comparison performed asdescribed above, includes residues in the sequences compared that arenot identical but have similar properties (e.g. as a result ofconservative substitutions). The % value of positives is determined bythe fraction of residues scoring a positive value in the BLOSUM 62matrix divided by the total number of residues in the longer sequence,as defined above.

In a similar manner, “percent (%) nucleic acid sequence identity” isdefined as the percentage of nucleotides in a candidate sequence thatare identical with the nucleotides in the UCP4 coding sequence. Theidentity values can be generated by the BLASTN module of WU-BLAST-2 setto the default parameters, with overlap span and overlap fraction set to1 and 0.125, respectively.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the UCP4 naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” nucleic acid molecule encoding a UCP4 polypeptide is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the UCP4-encoding nucleic acid. An isolatedUCP4-encoding nucleic acid molecule is other than in the form or settingin which it is found in nature. Isolated nucleic acid moleculestherefore are distinguished from the UCP4-encoding nucleic acid moleculeas it exists in natural cells. However, an isolated nucleic acidmolecule encoding a UCP4 polypeptide includes UCP4-encoding nucleic acidmolecules contained in cells that ordinarily express UCP4 where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers single anti-UCP4 monoclonal antibodies (including agonist,antagonist, and neutralizing antibodies) and anti-UCP4 antibodycompositions with polyepitopic specificity. The term “monoclonalantibody” as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a UCP4 polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Active” or “activity” for the purposes herein refers to form(s) of UCP4which retain the biologic and/or immunologic activities of native ornaturally-occurring UCP4. A preferred activity is the ability to affectmitochondrial membrane potential in a way that results in an up- ordown-regulation of metabolic rate and/or heat production. One suchactivity includes the generation of proton leakage in mitochondrialmembrane that results in an increase in metabolic rate. The activity maybe measured or quantitated in vitro or in vivo.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological and/or immunological activity of a native UCP4 polypeptidedisclosed herein. In a similar manner, the term “agonist” is used in thebroadest sense and includes any molecule that mimics a biological and/orimmunological activity of a native UCP4 polypeptide disclosed herein.Suitable agonist or antagonist molecules specifically include agonist orantagonist antibodies or antibody fragments, immunoadhesins of UCP4polypeptides, or fragments or amino acid sequence variants of nativeUCP4 polypeptides.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cows, horses, sheep, pigs, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

II. Compositions and Methods of the Invention

A. Full-length UCP4

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas UCP4. In particular, cDNA encoding a UCP4 polypeptide has beenidentified and isolated, as disclosed in further detail in the Examplesbelow. For sake of simplicity, in the present specification the proteinencoded by DNA 77568-1626 as well as all further native homologues andvariants included in the foregoing definition of UCP4, will be referredto as “UCP4,” regardless of their origin or mode of preparation.

As disclosed in the Examples below, a clone DNA 77568-1626 has beendeposited with ATCC. The actual nucleotide sequence of the clone canreadily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the UCP4 herein, Applicants have identified what isbelieved to be the reading frame best identifiable with the sequenceinformation available at the time of filing.

Using the Megalign DNASTAR computer program (and algorithms andparameters in this software set by the manufacturer) (Oxford MolecularGroup, Inc.), it has been found that a full-length native sequence UCP4(shown in FIG. 1 and SEQ ID NO:1) has about 34% amino acid sequenceidentity with UCP3, about 33% amino acid sequence identity with UCP2,and about 29% amino acid sequence identity with UCP1. Accordingly, it ispresently believed that UCP4 disclosed in the present application is anewly identified member of the human uncoupling protein family and maypossess activity(s) and/or property(s) typical of that protein family,such as the ability to enhance or suppress metabolic rate by affectingmitochondrial membrane potential.

B. UCP4 Variants

In addition to the full-length native sequence UCP4 polypeptidesdescribed herein, it is contemplated that UCP4 variants can be prepared.UCP4 variants can be prepared by introducing appropriate nucleotidechanges into the UCP4 DNA, and/or by synthesis of the desired UCP4polypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the UCP4, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the native full-length sequence UCP4 or in various domainsof the UCP4 described herein, can be made, for example, using any of thetechniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the UCP4 that results in a change in the amino acidsequence of the UCP4 as compared with the native sequence UCP4.Optionally the variation is by substitution of at least one amino acidwith any other amino acid in one or more of the domains of the UCP4.Guidance in determining which amino acid residue may be inserted,substituted or deleted without adversely affecting the desired activitymay be found by comparing the sequence of the UCP4 with that ofhomologous known protein molecules and minimizing the number of aminoacid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of 1 to 5 amino acids. The variation allowed may be determined bysystematically making insertions, deletions or substitutions of aminoacids in the sequence and, if desired, testing the resulting variantsfor activity in assays known in the art or as described herein.

One embodiment of the invention is directed to UCP4 variants which arefragments of the full length UCP4. Preferably, such fragments retain adesired activity or property of the full length UCP4.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the UCP4 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of UCP4

Covalent modifications of UCP4 are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of a UCP4 polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or the N- orC-terminal residues of the UCP4. Derivatization with bifunctional agentsis useful, for instance, for crosslinking UCP4 to a water-insolublesupport matrix or surface for use in the method for purifying anti-UCP4antibodies, and vice-versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazo-acetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis-(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)-dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the UCP4 polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence UCP4 (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequenceUCP4. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to the UCP4 polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence UCP4 (for O-linkedglycosylation sites). The UCP4 amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the UCP4 polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theUCP4 polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the UCP4 polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of UCP4 comprises linking the UCP4polypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The UCP4 of the present invention may also be modified in a way to forma chimeric molecule comprising UCP4 fused to another, heterologouspolypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of theUCP4 with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the UCP4. The presence ofsuch epitope-tagged forms of the UCP4 can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe UCP4 to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the UCP4 with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a UCP4 polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

The UCP4 of the invention may also be modified in a way to form achimeric molecule comprising UCP4 fused to a leucine zipper. Variousleucine zipper polypeptides have been described in the art. See, e.g.,Landschulz et al., Science, 240:1759 (1988); WO 94/10308; Hoppe et al.,FEBS Letters, 344:1991 (1994); Maniatis et al., Nature, 341:24 (1989).Those skilled in the art will appreciate that the leucine zipper may befused at either the 5′ or 3′ end of the UCP4 molecule.

D. Preparation of UCP4

The description below relates primarily to production of UCP4 byculturing cells transformed or transfected with a vector containing UCP4nucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare UCP4. Forinstance, the UCP4 sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of theUCP4 may be chemically synthesized separately and combined usingchemical or enzymatic methods to produce the full-length UCP4.

1. Isolation of DNA Encoding UCP4

DNA encoding UCP4 may be obtained from a cDNA library prepared fromtissue believed to possess the UCP4 mRNA and to express it at adetectable level. Accordingly, human UCP4 DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The UCP4-encoding gene may also be obtainedfrom a genomic library or by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to the UCP4 oroligonucleotides of at least about 20-80 bases) designed to identify thegene of interest or the protein encoded by it. Screening the cDNA orgenomic library with the selected probe may be conducted using standardprocedures, such as described in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).An alternative means to isolate the gene encoding UCP4 is to use PCRmethodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra, and are described above in SectionI.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined through sequence alignment using publicly available computersoftware programs (set to default parameters) such as BLAST, BLAST2,ALIGN, DNAstar, and INHERIT to measure identity or positives for thesequence comparison.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein, and, if necessary, using conventional primer extensionprocedures as described in Sambrook et al., supra, to detect precursorsand processing intermediates of mRNA that may not have beenreverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for UCP4 production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Depending on the host cell used,transformation is performed using standard techniques appropriate tosuch cells. The calcium treatment employing calcium chloride, asdescribed in Sambrook et al., supra, or electroporation is generallyused for prokaryotes or other cells that contain substantial cell-wallbarriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forUCP4-encoding vectors. Saccharomyces cerevisiae is a commonly used lowereukaryotic host microorganism.

Suitable host cells for the expression of glycosylated UCP4 are derivedfrom multicellular organisms. Examples of invertebrate cells includeinsect cells such as Drosophila S2 and Spodoptera Sf9, as well as plantcells. Examples of useful mammalian host cell lines include Chinesehamster ovary (CHO) and COS cells. More specific examples include monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinesehamster ovary cells/-DHFR(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human livercells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCCCCL51). The selection of the appropriate host cell is deemed to bewithin the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding UCP4 may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The UCP4 may be produced recombinantly not only directly, but also as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe UCP4-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), orthe signal described in WO 90/13646 published 15 Nov. 1990. In mammaliancell expression, mammalian signal sequences may be used to directsecretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2 μm plasmid origin is suitable for yeast,and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) areuseful for cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up theUCP4-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene, present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the UCP4-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding UCP4.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Req., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phospho-fructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

UCP4 transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the UCP4 by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theUCP4 coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding UCP4.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of UCP4 in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceUCP4 polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to UCP4DNA and encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of UCP4 may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of UCP4 can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify UCP4 from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of theUCP4. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology, 182 (1990); Scopes, Protein PurificationPrinciples and Practice, Springer-Verlag, New York (1982). Thepurification step(s) selected will depend, for example, on the nature ofthe production process used and the particular UCP4 produced.

E. Uses for UCP4

Nucleotide sequences (or their complement) encoding UCP4 have variousapplications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. UCP4 nucleic acid will also beuseful for the preparation of UCP4 polypeptides by the recombinanttechniques described herein.

The full-length native sequence UCP4 gene (described in Example 1; SEQID NO:2), or fragments thereof, may be used as, among other things,hybridization probes for a cDNA library to isolate the full-length UCP4gene or to isolate still other genes (for instance, those encodingnaturally-occurring variants of UCP4 or UCP4 from other species) whichhave a desired sequence identity to the UCP4 sequence disclosed in FIG.1 (SEQ ID NO:1). Optionally, the length of the probes will be about 20to about 80 bases. The hybridization probes may be derived from thenucleotide sequence of SEQ ID NO:2 or from genomic sequences includingpromoters, enhancer elements and introns of native sequence UCP4. By wayof example, a screening method will comprise isolating the coding regionof the UCP4 gene using the known DNA sequence to synthesize a selectedprobe of about 40 bases. Hybridization probes may be labeled by avariety of labels, including radionucleotides such as ³²P or ³⁵S, orenzymatic labels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the UCP4 gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

Fragments of UCP4 DNA contemplated by the invention include sequencescomprising at least about 20 to 30 consecutive nucleotides of the DNA ofSEQ ID NO:2. Preferably, such sequences comprise at least about 50consecutive nucleotides of the DNA of SEQ ID NO:2.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related UCP4 coding sequences.

Nucleotide sequences encoding a UCP4 can also be used to constructhybridization probes for mapping the gene which encodes that UCP4 andfor the genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for UCP4 encode a protein which binds toanother protein, the UCP4 can be used in assays to identify the otherproteins or molecules involved in the binding interaction. By suchmethods, inhibitors of the receptor/ligand binding interaction can beidentified. Proteins involved in such binding interactions can also beused to screen for peptide or small molecule inhibitors or agonists ofthe binding interaction. Also, the receptor UCP4 can be used to isolatecorrelative ligand(s). Screening assays can be designed to find leadcompounds that mimic the biological activity of a native UCP4 or areceptor for UCP4. Such screening assays will include assays amenable tohigh-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Small molecules contemplated include synthetic organic or inorganiccompounds. The assays can be performed in a variety of formats,including protein-protein binding assays, biochemical screening assays,immunoassays and cell based assays, which are well characterized in theart.

Nucleic acids which encode UCP4 or its modified forms can also be usedto generate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding UCP4 can be used to clone genomic DNA encodingUCP4 in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding UCP4. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for UCP4 transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding UCP4 introduced into the germline of the animal at an embryonic stage can be used to examine theeffect of increased expression of DNA encoding UCP4. Such animals can beused as tester animals for reagents thought to confer protection from,for example, pathological conditions associated with its overexpressionor underexpression. In accordance with this facet of the invention, ananimal is treated with the reagent and a reduced incidence of thepathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition.

Alternatively, non-human homologues of UCP4 can be used to construct aUCP4 “knock out” animal which has a defective or altered gene encodingUCP4 as a result of homologous recombination between the endogenous geneencoding UCP4 and altered genomic DNA encoding UCP4 introduced into anembryonic cell of the animal. For example, cDNA encoding UCP4 can beused to clone genomic DNA encoding UCP4 in accordance with establishedtechniques. A portion of the genomic DNA encoding UCP4 can be deleted orreplaced with another gene, such as a gene encoding a selectable markerwhich can be used to monitor integration. Typically, several kilobasesof unaltered flanking DNA (both at the 5′ and 3′ ends) are included inthe vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for adescription of homologous recombination vectors]. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected [see e.g., Li et al., Cell, 69:915(1992)]. The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras [see e.g.,Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of the UCP4polypeptide.

Nucleic acid encoding the UCP4 polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83, 4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

It is believed that the UCP4 gene therapy has applications in, forinstance, treating metabolic conditions. This can be accomplished, forexample, using the techniques described above and by introducing a viralvector containing a UCP4 gene into certain tissues (like muscle or fat)to increase metabolic rate in these targeted tissues and thereby elevateenergy expenditure.

Generally, methods of treatment employing UCP4 are contemplated by theinvention. Fuel combustion, electron transport, proton pumping and O₂consumption (which may be referred to collectively as metabolic rate)are coupled to ATP synthesis. There can be an “inefficiency” in mammals,such that a portion of metabolic rate (in some cases which may begreater than 20%) may be ascribed to H⁺ “leak” back into the matrixspace with no ATP synthesis.

It is believed UCP4 may be involved in catalyzing H⁺ leak, therebyplaying a role in energetic inefficiency in vivo. Accordingly,modulating UCP4 activity or quantities (presence) of UCP4 in mammaliantissues (particularly, metabolically important tissues), mayconcomitantly modulate H⁺ leak, metabolic rate and heat production. Themethods of modulating (either in an up-regulation or down-regulationmode) metabolic rate in a mammal has a variety of therapeuticapplications, including treatment of obesity and the symptoms associatedwith stroke, trauma (such as burn trauma), sepsis and infection.

In the treatment of obesity, those skilled in the art will appreciatethat the modulation of mitochondrial membrane potential may be used toincrease body metabolic rate, thereby enhancing an individual's abilityfor weight loss. Screening assays may be conducted to identify moleculeswhich can up-regulate expression or activity (such as the uncoupling) ofUCP4. The molecules thus identified can then be employed to increasemetabolic rate and enhance weight loss. The UCP4 polypeptides are usefulin assays for identifying lead compounds for therapeutically activeagents that modulate expression or activity of UCP4. Candidate moleculesor compounds may be assayed with the mammals' cells or tissues todetermine the effect(s) of the candidate molecule or compound on UCP4expression or activity. Such screening assays may be amenable tohigh-throughput screening of chemical libraries, and are particularlysuitable for identifying small molecule drug candidates. Small moleculesinclude but are not limited to synthetic organic or inorganic compounds.The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, cell based assays, etc. Such assay formats are well knownin the art.

Accordingly, in one embodiment, there is provided a method of conductinga screening assay to identify a molecule which enhances or up-regulatesexpression of UCP4, comprising the steps of exposing a mammalian cell ortissue sample believed to comprise UCP4 to a candidate molecule andsubsequently analyzing expression of UCP4 in said sample. In thismethod, the sample may be further analyzed for mitochondrial membranepotential. Optionally, the UCP4 is a polypeptide comprising amino acidresidues 1 to 323 of FIG. 1 (SEQ ID NO:1). The sample being analyzed maycomprise various mammalian cells or tissues, including but not limitedto human brain tissue. The candidate molecule employed in the screeningassay may be a small molecule comprising a synthetic organic orinorganic compound. In an alternative embodiment, the screening assay isconducted to identify a molecule which decreases or down-regulatesexpression of UCP4. The effect(s) that such candidate molecule may haveon the expression and/or activity or UCP4 may be compared to a controlor reference sample, such as for instance, expression or activity ofUCP4 observed in a like mammal.

UCP4 may also be employed in diagnostic methods. For example, thepresence or absence of UCP4, or alternatively over- or under-expressionof UCP4 in an individual's cells or tissues, can be detected usingassays known in the art, including those described in the Examplesbelow. Thus, the invention also provides a method of detectingexpression of UCP4 in a mammalian cell or tissue sample, comprisingcontacting a mammalian cell or tissue sample with a DNA probe andanalyzing expression of UCP4 mRNA transcript in said sample. The samplemay comprise various mammalian cells or tissues, including but notlimited to, human brain tissue. The skilled practitioner may useinformation resulting from such detection assays to assist in predictingmetabolic conditions or risk for onset of obesity. If it is determined,for instance, that UCP4 activity in a patient is abnormally high or low,therapy, such as hormone therapy, could be administered to return theUCP4 activity to a physiologically acceptable state.

Detection of impaired UCP4 function in the mammal may also be used toassist in diagnosing impaired neural activity or neural degeneration. Itis presently believed UCP4 may be involved in the regulation of braintemperature or metabolic rate that is required for normal brain function(and associated neural activity). It is also presently believed thatUCP4 may control the generation of reactive oxygen species and thereforecontribute to neural degeneration. Molecules identified in the screeningassays which have been found to suppress UCP4 expression or function mayalso be employed to treat fever since it is believed that UCP4 isup-regulated during episodes of fever.

F. Anti-UCP4 Antibodies

The present invention further provides anti-UCP4 antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-UCP4 antibodies may comprise polyclonal antibodies.

Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. The immunizing agent may include the UCP4 polypeptide or afusion protein thereof. It may be useful to conjugate the immunizingagent to a protein known to be immunogenic in the mammal beingimmunized. Examples of such immunogenic proteins include but are notlimited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor.

Examples of adjuvants which may be employed include Freund's completeadjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetictrehalose dicorynomycolate). The immunization protocol may be selectedby one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-UCP4 antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the UCP4 polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against UCP4.Preferably, the binding specificity of monoclonal antibodies produced bythe hybridoma cells is determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or'affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Human and Humanized Antibodies

The anti-UCP4 antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′), or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe UCP4, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

G. Uses for Anti-UCP4 Antibodies

The anti-UCP4 antibodies of the invention have various utilities. Forexample, anti-UCP4 antibodies may be used in diagnostic assays for UCP4,e.g., detecting its expression in specific cells or tissues. Variousdiagnostic assay techniques known in the art may be used, such ascompetitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-UCP4 antibodies also are useful for the affinity purification ofUCP4 from recombinant cell culture or natural sources. In this process,the antibodies against UCP4 are immobilized on a suitable support, sucha Sephadex resin or filter paper, using methods well known in the art.The immobilized antibody then is contacted with a sample containing theUCP4 to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the UCP4, which is bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the UCP4 from the antibody.

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The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Isolation of cDNA Clones Encoding Human UCP4

EST databases, which included public EST databases (e.g., GenBank), anda proprietary EST database (LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.), were searched for sequences having homologies to human UCP3.The search was performed using the computer program BLAST or BLAST2[Altschul et al., Methods in Enzymology, 266:460-480 (1996)] as acomparison of the UCP3 protein sequences to a 6 frame translation of theEST sequences. Those comparisons resulting in a BLAST score of 70 (or insome cases, 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the programAssemblyLIGN and MacVector (Oxford Molecular Group, Inc.).

A DNA sequence (“from DNA”) was assembled relative to other ESTsequences using AssemblyLIGN software (FIG. 7; SEQ ID NO:5). ESTs fromthe Incyte database included the sequences having the followingaccession nos.: 3468504; 3369262; 4220747; 1254733; 5016160; 3770189;2265329; 928717; 3715961; 3528102; 961523; 1863723; 382533; 918252;918404; 4313009; 3801604; c-swh06; 3464955; c-1sh09; 090424; 1316891;1342069; 1435593; 16014011; 1668098; 1668103; 222248; 243244; 246984;272663; 305678; 305871; 3369262; 3464955; and 3715961. In addition, thefrom DNA sequence was extended using repeated cycles of BLAST andAssemblyLIGN to extend the sequence as far as possible using the sourcesof EST sequences discussed above.

Based on this DNA sequence, oligonucleotides were synthesized to isolatea clone of the full-length coding sequences for UCP4 by PCR. Forward andreverse PCR primers generally range from 20 to 30 nucleotides and areoften designed to give a PCR product of about 100-1000 by in length. Theprobe sequences are typically 40-55 by in length. In some cases,additional oligonucleotides are synthesized when the consensus sequenceis greater than about 1-1.5 kbp.

PCR primers (forward and reverse) were synthesized:

forward PCR primer (SEQ ID NO: 3) CGCGGATCCCGTTATCGTCTTGCGCTACTGC (U401)reverse PCR primer (SEQ ID NO: 4)GCGGAATTCTTAAAATGGACTGACTCCACTCATC (U406)

UCP4 with an NH₂-terminal Flag-tag also was cloned into pcDNA3(pcDNA3Flag-UCP4; Invitrogen) between BamHI and EcoRI restriction sites.The following forward and reverse PCR primers were synthesized.

forward PCR primer (SEQ ID NO: 6) CGCGGATCCGAAATGGACTACAAGGACGACGATGACAAGTCCGTCCCGGAGGAGGAGG (U410) reverse PCR primer (SEQ ID NO: 4)GCGGAATTCTTAAAATGGACTGACTCCACTCATC (U406)

RNA for construction of the cDNA libraries was isolated from braintissue. The cDNA libraries used to isolated the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5E is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clone isolated by PCR as described above gave thefull-length DNA sequence for UCP4 (designated herein as DNA 77568-1626[FIG. 2, SEQ ID NO: 2] and the derived protein sequence for UCP4.

The entire coding sequence of UCP4 is shown in FIG. 2 (SEQ ID NO:2).Clone DNA 77568-1626 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 40-42,and an apparent stop codon at nucleotide positions 1009-1011. (See FIG.2; SEQ ID NO:2). The predicted polypeptide precursor is 323 amino acidslong. It is presently believed that UCP4 is a membrane-bound protein andcontains at least 6 transmembrane regions. These putative transmembraneregions in the UCP4 amino acid sequence are illustrated in FIG. 3. CloneDNA 77568, designated as DNA 77568-1626, contained in the pcDNA3 vector(Invitrogen) has been deposited with ATCC and is assigned ATCC depositno. 203134. UCP4 polypeptide is obtained or obtainable by expressing themolecule encoded by the cDNA insert of the deposited ATCC 203134 vector.Digestion of the deposited ATCC 203134 vector with BamHI and EcoRIrestriction enzymes will yield an approximate 972 plus 34 bp insert. Thefull-length UCP4 protein shown in FIG. 1 has an estimated molecularweight of about 36,061 daltons and a pI of about 9.28.

An alignment of the amino acid sequence of UCP4 with UCPs 1, 2 and 3 isillustrated in FIG. 3. Some notable differences were identified betweenUCP1 and UCP4. When UCP1 lacks its putative nucleotide binding site, itis resistant to inhibition by nucleotides, and when Phe-267 in UCP1 issubstituted with a Tyr residue, UCP1 has enhanced uncoupling activity.[Gonzalez-Barroso et al., Eur. J. Biochem., 239: 445-450 (1996);Mayinger et al., Biochem., 31: 10536-10543 (1992)]. Yet, like UCP2 andUCP3, UCP4 has a Tyr residue at this position. (See FIG. 3).Additionally, the carboxy-terminus of UCP1 has been implicated in theactivation of its uncoupling activity by free fatty acids (FFA).Substitution of Cys-305 by Ala or Ser residues results in eitherdecreased or increased activation by FFA, respectively.[Gonzalez-Barroso et al., supra]. Because UCP2 has an Ala-307, UCP3 hasa Ser-298, and UCP4 has a Ser-321, the uncoupling activity of UCP4 andthe other UCPs is likely regulated differently by nucleotides and FFA.

The human UCP4 gene has been mapped to chromosomal location 6 p11.2-q12which is closest to genomic marker SHGC-34952.

Example 2 Northern Blot Analysis

Expression of UCP4 mRNA in human tissues was examined by Northern blotanalysis. Human RNA blots were hybridized to a 1 kilobase ³²P-labelledDNA probe based on the full length UCP4 cDNA; the probe was generated bydigesting pcDNA3UCP4 and purifying the UCP4 cDNA insert. Human adult RNAblot MTN-II (Clontech) (FIGS. 4A, 4B, 4D, 4E, and 4F), human fetaltissue blot (FIGS. 4D and 4H), PBLs (FIGS. 4B and 4D), and cancer cells(FIG. 4C) were incubated with the DNA probes. As shown in FIG. 4C, thecancer cells probed included HL-60 (promyelocytic leukemia), HeLa cells,K562 (chronic myelogenous leukemia), MOLT-4 (lymphoblastic leukemia),Raji (Burkitt's lymphoma), SW480 (colorectal adenocarcinoma), A549 (lungcarcinoma), and G361 (melanoma). The expression of UCP2 was alsoexamined by probing a human brain multiple tissue blot with human UCP2cDNA. (FIG. 4G). All blots were subsequently probed with a β-actin cDNA.

Northern analysis was performed according to manufacturer's instructions(Clontech). The blots were developed after overnight exposure to x-rayfilm.

As shown in FIGS. 4A-4H, UCP4 mRNA transcripts were detected. Expressionwas seen in brain tissues, spinal cord, medulla, corpus callosum, andsubstantia nigra, but not in the other human tissues or cancer celllines examined. Although the UCP4 transcript level was higher in braintissues than in the spinal cord, medulla, corpus callosum, andsubstantia nigra (FIGS. 4A, 4E, and 4F), the UCP2 transcript levels werehigher in the spinal cord and medulla (FIG. 4G). In the human fetaltissue blot, the UCP4 transcript was only detected in the brain. (FIG.4H).

Example 3 Use of UCP4 as a Hybridization Probe

The following method describes use of a nucleotide sequence encodingUCP4 as a hybridization probe.

DNA comprising the coding sequence of full-length or mature UCP4 (asshown in FIG. 2, SEQ ID NO:2) is employed as a probe to screen forhomologous DNAs (such as those encoding naturally-occurring variants ofUCP4) in human tissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled UCP4-derived probe to the filters is performed in asolution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate,50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution, and 10% dextransulfate at 42° C. for 20 hours. Washing of the filters is performed inan aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence UCP4 can then be identified using standardtechniques known in the art.

Example 4 Expression of UCP4 in E. coli

This example illustrates preparation of UCP4 by recombinant expressionin E. coli.

The DNA sequence encoding UCP4 (SEQ ID NO:2) is initially amplifiedusing selected PCR primers. The primers should contain restrictionenzyme sites which correspond to the restriction enzyme sites on theselected expression vector. A variety of expression vectors may beemployed. An example of a suitable vector is pBR322 (derived from E.coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes forampicillin and tetracycline resistance. The vector is digested withrestriction enzyme and dephosphorylated. The PCR amplified sequences arethen ligated into the vector. The vector will optionally includesequences which encode for an antibiotic resistance gene, a trppromoter, a polyhis leader (including the first six STII codons, polyhissequence, and enterokinase cleavage site), the UCP4 coding region,lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. If no signal sequence is present, and theexpressed UCP4 is intracellular, the cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized UCP4 protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein. If a signal sequence is present, the expressed UCP4 can beobtained from the cell's periplasm or culture medium. Extraction and/orsolubilization of the UCP4 polypeptides can be performed using agentsand techniques known in the art. (See e.g. U.S. Pat. Nos. 5,663,304;5,407,810).

Example 5 Expression of UCP4 in Mammalian Cells

This example illustrates preparation of UCP4 by recombinant expressionin mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the UCP4 DNA is ligated into pRK5with selected restriction enzymes to allow insertion of the UCP4 DNAusing ligation methods such as described in Sambrook et al., supra. Theresulting vector is called pRK5-UCP4.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CRL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5-UCP4 DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspirated,off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of UCP4 polypeptide. The cultures containing transfected cellsmay undergo further incubation (in serum free medium) and the medium istested in selected bioassays.

In an alternative technique, UCP4 may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-UCP4 DNA is added.The cells are first concentrated from the spinner flask bycentrifugation and washed with PBS. The DNA-dextran precipitate isincubated on the cell pellet for four hours. The cells are treated with20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing expressed UCP4 can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

In another embodiment, UCP4 can be expressed in CHO cells. The pRK5-UCP4can be transfected into CHO cells using known reagents such as CaPO₄ orDEAE-dextran. As described above, the cell cultures can be incubated,and the medium replaced with culture medium (alone) or medium containinga radiolabel such as ³⁵S-methionine. After determining the presence ofUCP4 polypeptide, the culture medium may be replaced with serum freemedium. Preferably, the cultures are incubated for about 6 days, andthen the conditioned medium is harvested. The medium containing theexpressed UCP4 can then be concentrated and purified by any selectedmethod.

Epitope-tagged UCP4 may also be expressed in host CHO cells. The UCP4may be subcloned out of the pRK5 vector. The subclone insert can undergoPCR to fuse in frame with a selected epitope tag such as a poly-his taginto a Baculovirus expression vector. The poly-his tagged UCP4 insertcan then be subcloned into a SV40 driven vector containing a selectionmarker such as DHFR for selection of stable clones. Finally, the CH0cells can be transfected (as described above) with the SV40 drivenvector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedUCP4 can then be concentrated and purified by any selected method, suchas by Ni²⁺-chelate affinity chromatography.

In an alternative method, the UCP4 may be expressed intracellularly(where no signal sequence is employed). This intracellular expression,and subsequent extraction or solubilization and purification may beperformed using techniques and reagents known in the art.

Example 6 Expression of UCP4 in Yeast

The following method describes recombinant expression of UCP4 in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of UCP4 from the ADH2/GAPDH promoter. DNAencoding UCP4 and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof UCP4. For secretion, DNA encoding UCP4 can be cloned into theselected plasmid, together with DNA encoding the ADH2/GAPDH promoter, anative UCP4 signal peptide or other mammalian signal peptide, or, forexample, a yeast alpha-factor or invertase secretory signal/leadersequence, and linker sequences (if needed) for expression of UCP4.Alternatively, the native signal sequence of UCP4 is employed.

Yeast cells, such as S. cerevisiae yeast strain AB110, can then betransformed with the expression plasmids described above and cultured inselected fermentation media as set forth, for instance, in U.S. Pat.Nos. 4,775,662 and 5,010,00. The transformed yeast supernatants can beanalyzed by precipitation with 10% trichloroacetic acid and separationby SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant UCP4 can subsequently be isolated and purified by removingthe yeast cells from the fermentation medium by centrifugation and thenconcentrating the medium using selected cartridge filters. Theconcentrate containing UCP4 may further be purified using selectedcolumn chromatography resins. In an alternative method, the UCP4 may beexpressed intracellularly (where no signal sequence is employed). Theintracellular expression, and subsequent extraction or solubilizationand purification may be performed using techniques and reagents known inthe art.

Example 7 Expression of UCP4 in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of UCP4 inBaculovirus-infected insect cells.

The sequence coding for UCP4 is fused upstream of an epitope tagcontained within an expression vector. Such epitope tags includepoly-his tags and immunoglobulin tags (like Fc regions of IgG). Avariety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding UCP4 or the desired portion of the coding sequence ofUCP4 is amplified by PCR with primers complementary to the 5′ and 3′regions. The 5′ primer may incorporate flanking (selected) restrictionenzyme sites. The product is then digested with those selectedrestriction enzymes and subcloned into the expression vector. The vectormay contain the native signal sequence for UCP4 if secretion is desired.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged UCP4 can then be purified, for example, byNi²⁺-chelate affinity chromatography as follows. Extracts are preparedfrom recombinant virus-infected Sf9 cells as described by Rupert et al.,Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspendedin sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA;10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 secondson ice. The sonicates are cleared by centrifugation, and the supernatantis diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%glycerol, pH 7.8) and filtered through a 0.45 micron filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or Westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged UCP4 are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) UCP4 can beperformed using known chromatography techniques, including for instance,Protein A or protein G column chromatography.

Example 8 Measurement of Mitochondrial Membrane Potential Change Inducedby UCP4

Assays were conducted to determine the effects of UCP4 expression onmitochondrial membrane potential.

Human embryonic kidney 293 cells (ATCC CRL 1573) were grown in culturemedium (DMEM, 10% fetal bovine serum, 2 mM L-glutamine, 100 units/mlpenicillin, 100 microgram/ml streptomycin) to 60%-80% confluence in6-well plates and transiently transfected using FuGene™ 6 transfectionreagent (Boehringer Mannheim; according to manufacturer's instructions)with UCP-expressing constructs (pcDNA3UCP4 or pcDNA3UCP3),UCP-expressing constructs with a NH₂-terminal Flag-tag (pcDNA3Flag-UCP4or pcDNA3Flag-UCP3), or vector control (pcDNA3; available fromInvitrogen).

The expression constructs for cDNA encoding UCP4 with or without aNH₂-terminal Flag-tag were prepared according to Example 1. Expressionconstructs for cDNA encoding UCP3 were prepared by first obtaining cDNAencoding human UCP3 from a melanoma cDNA library by PCR. PCR primers(forward and reverse) were synthesized:

forward PCR primer (SEQ ID NO: 7)GCGAAGCTTGCCATGGTTGGACTGAAGCCTTCAGA (U301) reverse PCR primer(SEQ ID NO: 8) CGCGAATTCTCAAAACGGTGATTCCCGTAACAT (U302)

The expression construct for cDNA encoding UCP3 with a NH₂-terminalFlag-tag was prepared by the following PCR primers.

forward PCR primer (SEQ ID NO: 9)GCGAAGCTTGCCATGGACTACAAGGACGACGATGACAAG GTTGGACTGAAGCCTTCAGACG (U303)reverse PCR primer (SEQ ID NO: 8)CGCGAATTCTCAAAACGGTGATTCCCGTAACAT (U302)

UCP3 with or without the NH₂-terminal Flag-tag were cloned into pcDNA3(pcDNA3UCP3 and pcDNA3Flag-UCP3) between HindIII and EcoRI sites andconfirmed by DNA sequencing. Flag-tagged UCP3 and UCP4 expressed in 293cells were detected by Western blot analysis using anti-Flag M2monoclonal antibody (Kodak) and ECL detection kit (Pierce).

Mitochondrial membrane potential was analyzed according to methods knownin the art. [Salvioli et al., FEBS Lett., 411: 77-82 (1997); Smiley etal., Proc. Natl. Acad. Sci. USA, 88: 3671-3675 (1991)]. About 24-36hours post-transfection, cells were trypsinized, and 1.5×10⁶ werepelleted by centrifugation. The pelleted cells were resuspended in 0.5ml of a JC-1 dye solution and incubated in the presence or absence of 50μm CCCP (carbonylcyanide m-chlorophenylhydrazone; Sigma) in the dark for30 minutes at 37° C. JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanineiodide; Molecular Probes, Eugene, Oreg.) is a membrane potentialsensitive, fluorescent dye. To prepare the dye solution, JC-1 was firstprepared as a stock solution in dimethyl sulfoxide (DMSO; Sigma) at aconcentration of 5 mg/ml. The stock solution was diluted to 1 mg/ml withDMSO, and then further diluted to 10 μg/ml with culture medium prewarmedto 37° C. and filtered through both 0.45 μm and 0.2 μm filters toexclude aggregated JC-1.

The stained cells were washed and resuspended in 1.0 ml culture medium.The cells resuspended in culture medium were examined byspectrofluorometry (RF5000U Spectrofluorophotometer; SHIMADZU, Japan). Asubset of cells was analyzed by flow cytometry (Coulter EPICS Elite ESP,Hialeah, Fla.). For spectrofluorometric analysis, excitation was at 488nm and emission measured at 525 nm and 590 nm. Flow cytometry analysiswas performed with an argon laser of single 488 nm as excitation, afilter transmitting 525±20 nm in FL1 channel, and a filter transmittingabove 590 nm in FL2 channel. A minimum of 10,000 cells per sample wasanalyzed.

A statistical analysis was also performed. The mean ratios of red (593nm) versus green (532 nm) fluorescence intensity peaks fromspectrofluorometry were compared across treatments. There were nineindependent transfections per treatment. Differences were analyzed usingFisher's protected least significant difference.

The results are illustrated in FIGS. 5A and 5B. Expression of UCP3 inthe 293 cells reduced the fluorescent peak value ratio (593λ/532λ) byapproximately 15% (n=3) in comparison with that of the vector controltransfected cells, showing a decline in mitochondrial membranepotential. (FIG. 5A). In the cells transfected with UCP4, thefluorescence intensity indicative of membrane potential reductiondecreased by 19% (n=6) in comparison with that of the vector controltransfected cells. (FIGS. 5A and 5B). The NH₂-terminal Flag-tag had noeffect on the activity of UCP3 or UCP4.

A FACs analysis also showed a similar decline in mitochondrial membranepotential. In the FACs analysis, the integrated red-to-green intensityratios fell by 18% in UCP3-transfected cells and 24% in UCP4-transfectedcells. Cells treated with the chemical uncoupler, CCCP, also showed areduction of the red-to-green intensity ratio. (FIGS. 5A and 5B).

These data suggest that like UCP3, UCP4 has uncoupling activity.

Example 9 Preparation of Antibodies that Bind UCP4

This example illustrates preparation of monoclonal antibodies which canspecifically bind UCP4.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified UCP4, fusion proteins containing UCP4, andcells expressing recombinant UCP4 on the cell surface. Selection of theimmunogen can be made by the skilled artisan without undueexperimentation.

Mice, such as Balb/c, are immunized with the UCP4 immunogen emulsifiedin complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-UCP4 antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of UCP4. Three to four days later, the mice are sacrificed andthe spleen cells are harvested. The spleen cells are then fused (using35% polyethylene glycol) to a selected murine myeloma cell line such asP3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstUCP4. Determination of “positive” hybridoma cells secreting the desiredmonoclonal antibodies against UCP4 is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-UCP4monoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the to ascites can be accomplishedusing ammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 10 Subcellular Localization

To examine the subcellular location of UCP4, human breast carcinoma MCF7cells (ATCC HTB 22) were transfected with either pcDNA3Flag-UCP3(prepared according to Example 8) or pcDNA3Flag-UCP4 (prepared accordingto Example 1) using FuGene transfection reagent (Boehringer Mannheim).The transfected cells were fixed in 3% formaldehyde at room temperaturefor 15 minutes and permeabilized with 1% TritonX-100 for 15 minutes. Thecells were incubated with anti-Flag monoclonal antibody (10 μg/ml;Kodak) and anti-cytochrome C oxidase antibody (a mitochondrial marker)(3 ng/ml) for 20 minutes. The cells were then washed and incubated withCy3™-conjugated (donkey anti-mouse; Jackson Laboratories) andFITC-conjugated (donkey anti-rabbit, Jackson Laboratories) secondaryantibodies. The cells were then examined by fluorescence microscopy.

FIGS. 6A-6F show that UCP3 and UCP4 were co-localized with themitochondrial marker.

Example 11 The Expression of UCP4 mRNA in Mice Subjected to Food andTemperature Stresses

To evaluate whether UCP4 has uncoupling activity in situ important tometabolism, the amount of UCP4 mRNA produced in tissues of mice thatwere subjected to food and temperature stresses, i.e., metabolicchallenges, was determined. Depending on the role UCP4 may have inmetabolism, the amount of UCP4 mRNA produced in a tissue may vary withstresses to metabolism such as fasting, fat consumption, and exposure totemperatures below room temperature.

The mice in this study were fed normal rodent chow (Purina Rodent Chow5010; Purina, St. Louis, Mo.) and water ad libitum unless indicatedotherwise. The type of mouse studied varied depending on the conditionused to challenge the metabolism of the mouse studied and will bedescribed below.

Generally, the mice studied were exposed to light 12 hours a day from6:00 a.m. until 6:00 p.m. at which time they were exposed to dark forthe following 12 hours.

The mice were sacrificed under CO₂ just prior to tissue harvest, whichoccurred in the morning between 8:00 and 12:00 a.m. unless specifiedotherwise. The tissues were harvested and total tissue RNA was preparedusing reagents and protocols from Biotecx Lab, Houston, Tex. Although anumber of tissues were collected from each mouse, the study focused onmeasuring the abundance of UCP4 mRNA in the brain (because the brain hashigh UCP4 gene expression). At least 5 mice/treatment were used in thestudies.

Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR)was used to determine the amount of UCP4 mRNA in the harvested tissues.RT-PCR was performed using mRNA samples. [Heid et al., Genome Research,6:986-994 (1996); Gibson et al., Genome Research, 6:995-1001 (1996)].Generally, to carry out quantitative RT-PCR, primers and probes specificto UCP4 were used (TaqMan Instrument, PE Biosciences, Foster City,Calif.). Values were corrected for mRNA loading using β-actin mRNAabundance as loading control. The following primers and probes wereused:

For UCP4: (SEQ ID NO: 10) forward primer: 5′AAT GCC TAT CGC CGA GGA G3′;(SEQ ID NO: 11) reverse primer: 5′GTA GGA ACT TGC TCG TCC GG3′;(SEQ ID NO: 12) probe: 5′(FAM)TGC TCG CGC TCA CGC AGA GAT G (TAMARA)3′.For beta-actin: (SEQ ID NO: 13)forward primer: 5′GAA ATC GTG CGT GAC ATC AAA  GAG3′; (SEQ ID NO: 14)reverse primer: 5′CTC CTT CTG CAT CCT GTC AGC AA3′; (SEQ ID NO: 15)probe: 5′(FAM)CGG TTC CGA TGC CCT GAG GCT C (TAMARA)3′.The Effect of Food Consumption on UCP4 mRNA Expression

In a first study, seven-week old male mice (C57BL/6J; Bar Harbor, Me.)were studied to evaluate the effect of fasting and eating meals on UCP4mRNA production in the mice studied. The mice were obtained at six weeksof age and at seven weeks were randomly assigned to one of three groups:control mice fed ad lib, mice fasted for 24 hours, and mice fasted for24 hours and then fed ad lib for 24 hours.

The mice were sacrificed as described above after ad lib feeding for thefirst group, after 24 hours of fasting for the second group, and afterthe 48 hours of first fasting and then ad lib feeding for the thirdgroup. The tissues were harvested as described above.

Quantitative RT-PCR was performed for the brain tissue according to themethods described above and the amount of UCP4 mRNA produced in thebrain was quantified. Statistical differences across the groups weredetermined using a protected Fisher's least significant differenceanalysis (L. Ott, An Introduction to Statistical Methods and DataAnalysis, 3rd Ed., Boston: PWS-Kent Publishing Co., 1988). The datapresented in FIGS. 8A to 8C represent means+/−SEM.

FIG. 8A illustrates the UCP4 mRNA abundance in the brain tissue frommice that were fed ad lib for 24 hours. FIG. 8B illustrates the UCP4mRNA abundance in the brain tissue from mice that fasted. FIG. 8Cillustrates the UCP4 mRNA abundance in the brain tissue from mice thatfasted for 24 hours and then were fed ad lib for 24 hours.

Typically, fasting and restriction of food consumption decreasemetabolic rate, suggesting that expression of UCP4 mRNA would decreasefor mice that were fasting compared to mice that were fed ad lib. YetFIG. 8B does not show a decrease in UCP4 mRNA expression in brain tissuefor the mice that fasted compared to the mice that were fed ad lib asshown in FIG. 8A.

The Effect of Fat Consumption on UCP4 mRNA Expression

In a second study, four-week old male mice (A/J or C57BL/6J; JacksonLabs, Bar Harbor, Me.) were studied to evaluate the effect of high andlow fat diets on UCP4 mRNA production in the mice studied. A/J mice havebeen shown to be “obesity-resistant” on a high fat diet compared to“obesity-prone” C57BL6/J (see Surwit et al., supra). This may be due toa lower metabolic efficiency in the A/J strain—i.e., they apparently puton fewer calories per calories ingested.

The mice were obtained at four weeks of age and immediately placed oneither a low fat diet or high fat diet (Research Diets, Inc., NewBrunswick, N.J.) patterned after those formulated by Surwit et al.,Metabolism, 44(5): 645-651 (1995), containing 11% or 58% fat (%calories), respectively. Animals were fed ad lib for approximately threeweeks (days 22-23 on diet). They were then sacrificed, and their tissueswere harvested as described above. Quantitative RT-PCR was performed forthe brain tissue according to the methods described above and the amountof UCP4 mRNA produced in the brain tissue was quantified. Statisticaldifferences across the groups were determined using a protected Fisher'sleast significant difference analysis (L. Ott, An Introduction toStatistical Methods and Data Analysis, 3rd Ed., Boston: PWS-KentPublishing Co., 1988). The data presented in FIGS. 9A to 9D representmeans+/−SEM.

FIG. 9A illustrates the UCP4 mRNA abundance in brain tissue from A/Jmice that were fed a low fat diet, and FIG. 9B illustrates the UCP4 mRNAabundance in brain tissue from A/J mice that were fed a high fat diet.FIG. 9C illustrates the UCP4 mRNA abundance in brain tissue fromC57BL6/J mice that were fed a low fat diet, and FIG. 9D illustrates theUCP4 mRNA abundance in brain tissue from C57BL6/J mice that were fed ahigh fat diet.

The Effect of Temperature Stress on UCP4

In a third study, male mice (FVB-N; Taconic, Germantown, N.Y.) werestudied to evaluate the effect of exposing the mice to temperaturestresses. Typically, cold exposure in rodents elicits an increase inmetabolic rate. This metabolic increase may be to support a stable bodytemperature. Yet warm-acclimation, which is defined as chronic exposureto temperatures within the murine thermoneutral zone (approx. 30-35°C.), lowers metabolic rate. [Klaus et al., Am. J. Physiol.,274:R287-R293 (1998)].

The mice in this study were housed two per cage and were randomlyassigned to the following groups: a control group (housed at 22° C. for3 weeks), a warm-acclimated group (housed at 33° C. for 3 weeks), afood-restricted group (housed at 22° C. for 3 weeks but given accesseach day to the average amount of food eaten by warm-acclimated mice theday before), a cold-challenged group (housed at 22° C. for 3 weeks priorto the initiation of exposure to 4° C.). For the cold-challenged mice,beginning in the morning, mice were exposed to 4° C. by being placedinto a 4° C. room for 1, 6, 24, or 48 hours prior to sacrificing themice and harvesting the tissue.

The mice were sacrificed and tissues were harvested at six week of ageas described above. Quantitative RT-PCR was performed for the braintissue according to the methods described above and the amount of UCP4mRNA produced in the brain was quantified. Statistical differencesacross the groups were determined using a protected Fisher's leastsignificant difference analysis (L. Ott, An Introduction to StatisticalMethods and Data Analysis, 3rd Ed., Boston: PWS-Kent Publishing Co.,1988). The data presented in FIGS. 10A to 10G represent +/−SEM. Anasterisk indicates a statistical difference of at least p<0.05.

FIG. 10A illustrates the UCP4 mRNA abundance in the control group ofmice. FIGS. 10B to 10E illustrate the UCP4 mRNA abundance in the groupof mice that were cold-challenged for 1, 6, 24, and 48 hours,respectively. FIG. 10F illustrates the UCP4 mRNA abundance in thefood-restricted group of mice, and FIG. 10G illustrates the UCP4 mRNAabundance in the warm-acclimated group of mice.

FIGS. 10B through 10E all indicate an increase in UCP4 mRNA expressionin the cold-challenged mice compared to the control group shown in FIG.10A. FIGS. 10F and 10G do not show a similar increase in UCP4 mRNAexpression for the food-restricted mice and the warm-acclimated mice,respectively, compared to the control group shown in FIG. 10A.

* * * *

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,USA (ATCC):

Material ATCC Dep. No. Deposit Date DNA77568-1626 203134 Aug. 18, 1998

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC '122 and the Commissioner's rules pursuantthereto (including 37 CFR '1.14 with particular reference to 8860G 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An isolated UCP4 polypeptide comprising amino acid residues from 1 to323 of FIG. 1 (SEQ ID NO: 1).
 2. The isolated UCP4 polypeptide of claim1, wherein the UCP4 polypeptide is encoded by a cDNA insert of a vectordeposited as ATCC Deposit No. 203134 (DNA 77568-1626).
 3. The isolatedUCP4 polypeptide of claim 1 consisting essentially of amino acidresidues 1 to 323 of FIG. 1 (SEQ ID NO:1).
 4. The isolated UCP4polypeptide of claim 1 consisting of amino acid residues 1 to 323 ofFIG. 1 (SEQ ID NO:1).
 5. A process for producing a UCP4 polypeptidecomprising: (a) transforming a host cell with a vector comprising anisolated nucleic acid molecule comprising a DNA molecule encoding a UCP4polypeptide comprising the sequence of amino acid residues 1 to 323 ofFIG. 1 (SEQ ID NO: 1); (b) culturing the host cell under conditionssuitable for expression of a UCP4 polypeptide; and (c) recovering saidUCP4 polypeptide from the cell culture.
 6. The process of claim 5,wherein the vector is operably linked to control sequences recognized bythe host cell transformed with the vector.
 7. The process of claim 6,wherein the host cell is a CHO cell.